1. Field of the Invention
This invention relates to a method to analyze arachidonic acid (AA)-derived products which are immunoreactive with antibodies produced against hydroxyeicosatrienoic acids (DHETs). More specifically, the present invention relates to a method which can be used to facilitate investigations of the physiological and pathophysiological roles of the metabolic products of arachidonic acid epoxygenases and epoxide hydrolases. The present invention also relates to a method to assess catalytic activity of AA epoxygenases using and a method to decrease hepatic AA epoxygenase expression by treatment with glucocorticoids.
2. Description of Related Art
AA is a component of cellular membranes and plays a critical role as a mediator of cell and organ function through its metabolic cascade. The AA cascade includes prostaglandin synthases, lipoxygenases, and cytochromes P450 (CYPs). The CYP pathway is composed of lipoxygenases-like (allylic oxidation), xcfx89/xcfx89-1 oxygenases and epoxygenases (olefin epoxidation), which metabolize AA to produce 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs), 16- to 20-hydroxyeicosatetraenoic acids (OH-AAs), and 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), respectively (1). Epoxide hydrolases hydrolyze biologically active EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs).
CYPs which have been identified to be AA epoxygenases are CYP2C11, CYP2C23, CYP2B1, and CYP2B2 expressed in rats (1), CYP2C1 and CYP2C2 expressed in rabbits (2,3), and CYP2C8 and CYP2C9/2C10 expressed in human (4). Recently, CYP2J, a novel CYP subfamily which is abundantly expressed in extrahepatic tissues, has been found in humans (CYP2J2) (5), rats (CYP2J3) (6) and rabbits (CYP2J1) (7).
EETs are synthesized in many tissues including liver, brain, eye, adrenal gland, blood vessels, and kidney, and circulate in blood. EETs are excreted in urine (8-10). Recently, it has been reported that EETs and DHETs are also synthesized in the lung, heart and gastrointestinal tract (5,7,11).
EETs have potent vasoactive properties (vasodilator or vasoconstrictor), increase cytosolic Ca+ concentration and renal Na+ transport, and stimulate hormone release in many tissues including insulin and glucagon from pancreatic islet cells, growth horrnone, oxytocin and vasopressin from the pituitary gland, and catecholamines from adrenal gland (9). Recently, it is reported that 14,15-EET functions as a second messenger in epidermal growth factor-mediated signaling pathway (12).
Urinary excretion of Na+, EETs and DHETs decreased after inhibition of AA epoxygenase activity by treating rats with clotrimazole, which induced salt-sensitive and clotrimazole-dependent hypertension. A salt-sensitive phenotype of the Dahl rat was associated with a lack of increases in renal AA hypoxygenases after intake of a high salt diet (10,13). A spontaneous hypertensive rat (SHR) study indicated that xcfx89/xcfx89-1 hydroxylase activity of kidney microsomes was significantly higher than that of normotensive Witstar Kyoto rats (WKY) whereas AA epoxygenase activity (EETs+DHETs) showed no difference between two strains at any age group tested (14). Urine samples were not tested. Thus it is generally concluded that the developmental phase of hypertension was linked to increases in the activity of kidney microsomal xcfx89/xcfx89-1 hydroxylase. Indeed, recently the gene coding for CYP4A2 (xcfx89/xcfx89-1 hydroxylase) was found to be preferentially expressed in SHR (14,15).
So far, levels of urinary EETs or DHETs of SHR have not been measured or compared with those of WKY. Thus, our findings that DHET levels in urine specimens obtained from SHR is xcx9c56-fold higher than those of WKY and existence of free and conjugated DHETs and metabolites of DHETs in the SHR urine specimens were surprising. Our result strongly suggest that epoxide hydrolases expressed in kidney play a critical role in hypertension. Thus, measurement of total DHET levels in urine provides better correlation of AA epoxygenase-epoxide hydrolase activities with hypertension.
Levels of EETs in biological specimens, thus far, were measured after chemical hydrolysis to DHETs by high-performance liquid chromatography (HPLC) followed by negative-ion chemical ionization/gas chromatography/mass spectrometry (NICI/GC/MS). This method is not suitable for clinical routines because a) sample preparation for GC/MS is long and tedious, b) The method assesses only free EETs, EETs after chemical hydrolysis to DHETs, or DHETs and c) it is not practical to measure all the possible EET or DHET metabolites using GC/MS. EET levels in human urine samples and human urine specimens during pregnancy were assayed. However, the EET levels were measured after chemical hydrolysis to DHETs by HPLC/GC/MS (16,17). So, although the data is shown to be DHET, it is only measuring primarily EETs converted to DHET.
Recently free EETs (after chemical hydration to DHETs) were measured by using fluoroimmunoassay (FIA) and radioimmunoassay (RIA) to measure EET release from cells (18). However, again the EET levels of the cell medium extract were measured after chemical hydrolysis of the EETs to DHETs. No prior art reference directs one skilled in the art to the important relationship between the total (free and conjugated) metabolites of AA epoxygenases and epoxide hydrolases and hypertension, nor their measurement in urine samples.
According to the present invention, a method to assess hypertension mediated by AA epoxygenase and epoxide hydrolase by measuring the amount of metabolites of epoxygenases and epoxide hydrolases (DHETs) in a biological sample which contains the DHETs (using any methods including GC/MS or ELISA). The method further included determining the amount of DHET-specific epitopes immunoreactive with antibodies produced against DHETs present in the sample. This amount is compared with a control sample(s). The hypertension mediated by AA epoxygenase and epoxide hydrolase is determined through the comparison wherein the amount of increase in the free and conjugated DHETs and metabolites of DHETs in the sample isolated from an organism. The present invention also provides an immunoassay to assess catalytic activity of AA epoxygenases by subtracting the amounts of NADPH-independent EETs from total (NADPH-dependent+independent) EETs after incubation of the enzyme with NADPH. The present invention also provides a method to decrease hepatic AA epoxygenase expression by treatment with a glucocorticoid including dexamethasone.